PROJECT SUMMARY The goal of this study is to develop new strategies to expand human hematopoietic stem cells (HSC) by utilizing cryopreserved umbilical cord blood (CB) in NHLBI Biorepository. HSC transplantation is currently being used as regenerative medicine for the treatment of congenital deficiencies and malignant diseases of the blood and immune systems. A major roadblock of success of this therapy is the limited number of HSC per harvest and the poorly understood expansion and differentiation behavior. This proposal aims to increase CB HSC ex vivo expansion by targeting a newly identified molecular pathway. Natural genetic diversity offers an important yet largely untapped reservoir for deciphering molecular regulatory mechanisms of HSCs and hematopoiesis. We previously identified the first and probably so far the only stem cell regulatory gene, latexin (Lxn), by employing genetic diversity approach. Lxn is a negative regulator of HSC number and function. Our preliminary work in Lxn knockout mice showed that Lxn inactivation increases HSC numbers, and the expansion is controlled within the physiological range of variation. The expanded HSCs maintain the capacity of self-renewal and multilineage differentiation. Considering the nature of Lxn as a natural variant regulator of HSCs, we speculate that Lxn may function similarly in human population. Our preliminary studies demonstrate the negative correlation between HSC numbers and Lxn expression in human samples. We therefore hypothesize that Lxn negatively regulates human HSCs, and its inhibition leads to HSC expansion by increasing self-renewal while maintaining multi-lineage differentiation capacity. Specific aims are to: 1) determine whether knockdown of Lxn can expand human HSCs while maintaining their self-renewal and multilineage differentiation capacity, and 2) Determine the molecular mechanisms by which Lxn-Thbs1 signaling pathway regulates human HSC expansion and function. This research will provide new insight into the potential utility of Lxn inhibition to promote HSC expansion, thereby benefiting patients needing HSC transplantation.